Zubrycka, Agata
(2022)
Investigation of the biochemistry of post-translational modifications to Group VII ETHYLENE RESPONSE
FACTORS.
PhD thesis, University of Nottingham.
Abstract
In Arabidopsis thaliana the group VII ETHYLENE RESPONSE FACTOR (ERFVII) transcription factors (RAP2.12, RAP2.2, RAP2.3, HRE1, HRE2) have been shown to be substrates of the PLANT CYSTEINE OXIDASE (PCO) branch of PROTEOLYSIS (PRT6) N-degron pathway, that acts to regulate oxygen and nitric oxide sensing in plants. ERFVIIs share a conserved second position Cys motif, which is assumed to undergo posttranslational modifications in the presence of O2 and nitric oxide (NO). In the currently understood pathway Cys-2 is exposed following methionine cleavage by METHIONINE AMINOPEPTIDASES (MAPs) and undergoes oxidation by PCOs to Cys (SO2H), which allows subsequent arginylation by ARGININYL TRANSFERASEs (ATE). The amino terminal (Nt)-Arg residue is recognised by E3 ligase PRT6, leading to 26S proteasome degradation. The enzymatic components of the PCO N-degron pathway are oxygen and NO sensors and its substrates are transducers of the hypoxia or NO deficit. However, the exact chemical nature of Ntmodifications of pathway substrates, and in particular the oxidative state of Cys-2 are still under discussion and have not been investigated directly. In addition, NO affects the PCO N degron pathway at a yet to be identified point. Here the stability of ERFVIIs RAP2.3, HRE2 and HRE1 was studied in vivo in response to oxygen restricted conditions (submergence), in the presence of a 26S proteasome inhibitor (bortezomib) and in mutants of the PCO N-degron pathway. These analyses revealed an unexpectedly slower than predicted mobility of stable ERFVIIs during sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The possibility of post-translational modifications (PTMs) affecting the stability and size of RAP2.3 was investigated, including possible ubiquitin-like PTM addition. Studies of physiochemical properties related to amino acid composition responsible for the unexpected slower gel mobility were conducted in in vitro cell-free Wheat germ (WG) and Rabbit Reticulocyte (RR) lysates and with recombinant RAP2.3-6xHis produced in Escherichia coli. These analyses excluded the possibility of PTMs as a cause of the slower gel mobility of ERFVIIs. Results showed the direct influence of a highly hydrophobic amino acid composition in the C-terminal region of RAP2.3, on the gel mobility. The physiochemical reasons for this were investigated using bioinformatic tools.
The oxidation stated of Cys-2 was the second subject of research in this study. According to present knowledge, Cys-2 is oxidised to sulfinic acid (SO2H) before arginylation. However, in this study, using a plant extract in combination with Mass Spectrometry (MS) approaches, I observed formation of sufonic acid (SO3H) at Nt-Cys of RAP2.3 and RAP2.12 in addition to Nt-arginylation. The oxidative state of Cys-2 of RAP2.3 and RAP2.12 was investigated further using the plant extract in the presence of PCO N-degron pathway and mono-oxygenases inhibitors, in order to investigate state of Nt-Cys at each step of the PCO Ndegron pathway, as well as origin of the third oxygen incorporated into Nt-Cys-sulfonic acid. These analyses revealed that Nt-Cys oxidation and subsequent arginylation were inhibited by excessive Zn2+ concentration and 2,2′-Dipyridyl disulfide (DIP) iron chelator (inhibitors of PCO activity). Also, the possibility of incorporation of oxygen from water into Nt-Cys sulfinic acid of RAP2.3 was excluded by showing a lack of incorporation of oxygen from H218O. Monooxygenases inhibitors were used to identify the possible enzyme-type responsible for the third oxygen incorporation into Nt-Cys- of RAP2.3. Finally, the state of Nt-Cys was investigated in the presence of NO scavenger 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3- oxide potassium salt (cPTIO), and tannic acid (an ATE1 inhibitor), both of which revealed lower ratios of Arg-Cys-sulfonic acid, as well as for the first-time suggesting the possibility of MAP inhibition by cPTIO and tannic acid, which blocks PCO N-degron pathway activity by preventing Cys-2 exposure.
Importantly, this study changes the current paradigm regarding the processing of NtCys by showing that in vivo Nt-Cys is oxidised to Cys-sulfonic acid not the currently accepted view that sulfinic acid is the final oxidation state. This suggests the presence of addition monooxygenase activity, which would convert Cys-sulfinic acid to sulfonic or add one oxygen to Nt-Cys in order to facilitate incorporation of two oxygens by PCOs. Based on these observations, study of the degradation of PCO N-degron pathway substrates requires further investigation, aiming to identified yet unknown additional component(s) including additional mono-oxygenase activity involved in Nt-Cys oxidation. Identification of mono-oxygenase(s) responsible for the third oxygen incorporation might help with creating crops more resistant to flooding, drought or pathogens. In our times climate changes become more severe each year. Floods, drought, or sudden drop in temperature reduces plant immunity to pathogens. Thus, investigation of novel components of the PCO N-degron pathway might support crop breeding and selection in order to improved yield and could allowed plants to grown in places which due to climate changes couldn’t be their habitats.
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